Purification Process Development of Protein Subunit
based Vaccine Candidates Produced using
Recombinant E. coli Expression System-Case Study
Davinder Chawla and Yan-Ping Yang
Bio Process Research & Development, sanofi pasteur, Toronto
Part I
ABSTRACT
Results (2)
Proteins are key components of prophylactic vaccines against
infectious diseases. Protein subunit based vaccine is an attractive
alternative to the traditional detoxified bacterial or inactivated viral
vaccine approach due to its highly purified and well characterized
product nature. Purification of protein antigens to achieve consistent
product purity and quality is an integral part of the protein subunit
vaccine product development process. Expression levels of the
recombinant proteins in bacterial expression system may be
extremely high following rapid technology advancement. The
challenges and approaches used to develop purification processes for
novel protein vaccine candidates expressed at g/L level are being
discussed.
Introduction and Objective
•The concept of Platform Technology (capturing, purification and
polishing) has been successfully applied to the Mab purification
(Figure 1).
•The objective of this study was to assess whether a similar concept
can be applied to the purification of E. Coli expressed soluble
recombinant proteins in order to facilitate vaccine antigen purification
process development.
•A case study with a specific challenge will also be discussed (Part II)
Crude harvest
Target:
50% purity
Target:
70-80% purity
Target:
>90% purity
Methods
Capturing or gross
purification
Figure 1: Typical Purification
Process
Intermediate
purification
Polishing
Concentration / buffer
exchange
•Chromatographic methods such as Anion or cation exchange,
Hydrophobic interaction chromatography, mixed mode chromatography
methods were used.
•AKTA Explorer was used for small-scale method development and
processes were scaled-up using AKTA PILOT.
•In-Process samples were tested using inline monitoring by UV,
conductivity etc.
•Fractions were analyzed by SDS-PAGE or BCA protein assay.
Results (1)
•Several protein purification processes were developed using different
protein antigens expressed in E. coli expression systems.
•A trend was observed in most of these purification schemes i.e. strong
anion exchange such as Q column worked well as primary capture step
and had enough capacity to capture the target protein where the
expression in upstream was 1-3 g/L (Figure 1)
•Binding and elution conditions of each protein including the buffers to
purify these protein varied and were dependent on the individual
characteristics of these proteins (data not shown).
•The purity of in-process fractions after 1st unit operation was typically
>50%. (Figure 2)
KDa
250
150
100
75
50
37
25
20
15
10
Figure 2: SDS-PAGE Analysis of
Fractions from 1st unit operation
1= M.W. std
2= Standard
3= Crude
4= Flow
through
5=Intermediat
e wash step
6= Elution
Peak
7= Elution Tail
8= Strip
•The 2nd unit operation selected
was either a cation exchanger
(such as SP) or Hydrophobic
column chromatography (HIC)
step.
•The purity of in-process
samples after 2 unit operation
was typically >80% and for one
protein was even >90% (Figure
3).
•Binding and elution conditions
of each protein including the
buffers to purify these proteins
on cation exchanger or HIC
varied and were dependent on
the individual characteristics of
these proteins (data not shown).
KDa
250
150
100
75
50
37
25
20
15
10
Figure 3: SDS-PAGE Analysis of
Fractions from 2nd unit operation
1=M.W. Std
2=Standard
3=First column
purified
4=Flow Through
5= Elution
6. Elution Tail
7= Strip
Results (3)
•The 3rd unit operation selected was
almost always a mixed mode column
chromatography step such as
Ceramic Hydroxyapatite or Q
membrane. The purity of in-process
samples after 3 unit operation was
typically >95% and met the criteria
(Figure 4).
•Binding and elution conditions of
each protein including the buffers to
purify these proteins on 3rd column
were dependent on the individual
characteristics of these proteins
(data not shown).
•Three or two column purified
material was used for final buffer
exchange using Tangential Flow
Filtration.
KDa
250
150
100
75
50
37
25
20
15
10
Figure 4: SDS-PAGE
Analysis of Fractions
from 3rd unit operation
1= M.W. Std.
2=Standard
3= Two column
purified
4= Flow Through
5=Intermediate Wash
6-9=Elution
10-Strip
Conclusions
A common theme or the standardized approach can
be used to develop new purification processes for new
recombinant E. Coli antigens (Figure 5).
Crude harvest
Crude harvest
Target:
50% purity
Capturing or gross
purification
Anion exchange
Target:
70-80% purity
Intermediate
purification
Target:
>90% purity
Polishing
Concentration / buffer
exchange
Cation exchange, or
hydrophobic
Mixed mode or Anion
exchange or
membrane
Tangential Flow
Filtration (TFF)
Figure 5: Common theme in the purification of soluble
recombinant proteins in E. Coli